Treatment and prevention of injury due to radiation exposure

ABSTRACT

The disclosure relates to the treatment and/or prevention of radiation damage by administering a reverse micelle system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage of PCT/EP2019/068099, filed onJul. 5, 2019, which claims the benefit of U.S. Provisional ApplicationNo. 62/694,637, filed on Jul. 6, 2018, the disclosures of which arehereby incorporated by reference in their entireties.

FIELD OF INVENTION

The disclosure relates to the treatment and/or prevention of injury dueto radiation exposure by administering a reverse micelle system.

BACKGROUND

There have been significant advances in developing safe and effectiveradiation countermeasures for radiation injuries. However, only a fewagents have been approved by the U.S. Food and Drug Administration (FDA)for human use in a non-clinical setting (e.g., dirty bomb, mass nuclearaccident). For example, amifostine (Ethyol®) has not been approved bythe FDA as radioprotector for the treatment of acute radiation syndrome(ARS), but only for a limited clinical use for patients undergoingradiotherapy or chemotherapy. Furthermore, amifostine is effective onlywhen administered intravenously (i.v.) or subcutaneously (s.c.), and hasa short-time window of effectiveness, and thus is not adapted tomass-casualty situation.

Two granulocyte colony stimulating factors (G-CSF) (Neupogen® andNeulasta®) have been improved by the FDA as radiomitigators for thetreatment of the hematopoietic sub syndrome of ARS (H-ARS). But theserecombinant agents have limitations such as the need to be injected,multiple doses are required, and monitoring of side effects.

Several drugs are in different stages of evaluation by the FDA asradiation countermeasures for the prevention or treatment of ARS but sofar none possesses all the requisite qualities to be an optimumradiation countermeasure, e.g., low toxicity, wide window of protection,stability under extreme conditions, easy to handle and to administer.

Superoxide dismutases (SOD) are enzymes that catalyze the dismutation ofsuperoxide anions to oxygen and hydrogen peroxide and they are known forthe involvement in antioxidant defense mechanisms (Huang, 2012). As theclinical application of manganese superoxide dismutase (Mn-SOD) islimited because of its short half-life, high molecular weight, andinability to cross cell membrane freely, other strategies such as SODmimics have been developed (Miriyala, 2012). For example, AEOL10150, ametalloporphyrin Mn-SOD mimetic developed by Aeolus Pharmaceuticals,mitigates acute radiation-induced lung injury after multiple injections(MacVittie, 2017).

Greenberger and co-workers investigated a SOD-based gene therapystrategy. They showed that Mn-SOD-plasmid in liposomes intravenouslyadministered in rodents before ionizing radiation exposure has a potentefficacy to protect normal tissues (Epperly, 2008). In another studythey showed that the daily administration of a diet rich in a mixture ofmicronutrient multivitamin and trace element including manganese,antioxidants and chemopreventive agents and optionally a mixture offatty acids, does not ameliorate survival up to the thirty-day mark but,in animals surviving the acute effects of ionizing radiation, the dietameliorates the radiation exposure-induced life shortening (Epperly,2011 and US2014/0023701).

Murata and coworkers showed that manganese chloride, administeredintraperitoneally does not protect against acute radiation injury ofskin or crypt cells (Murata, 1995).

Manganese is not easy to administered due to its poor oralbioavailability and toxicity including progressive neurodegenerativedamages with an associated motor dysfunction syndrome similar to thatseen in Parkinson disease (Williams, 2012).

Several reverse micellar systems are known in the art. These systemsrequire specific components and/or are directed to uses other than thetreatment and/or prevention of radiation damage. For example, U.S. Pat.No. 9,592,218 discloses a reverse micelle system comprising at least onemetal ion, a sterol, an acylglycerol, lecithin, alcohol and water, andmethods for the treatment or improvement of symptoms of various diseasesor disorders, but does not disclose the treatment and/or prevention ofradiation damage.

U.S. Pat. No. 9,452,136 discloses a reverse micelle system comprising atleast one nucleic acid, a sterol, an acylglycerol, a phospholipid or asphingolipid, an alcohol and water, and methods for the treatment orimprovement of symptoms of various diseases or disorders.

U.S. Pub. No. 2017/0035909 discloses a reverse micellar systemcomprising at least a chelating or sequestering agent, an acylglycerol,lecithin, an alcohol and water, and methods of one disease linked to theaccumulation and/or overload of at least one radionuclide or metal.

WO 2017/005899 discloses the preparation of cyano-bridged metalnanoparticles within a biocompatible reverse micellar system. Thebiocompatible reverse micellar system or the cyano-bridged metalnanoparticles comprised therein are used as a contrast agent and/or adiagnosis agent. The biocompatible reverse micellar system or thecyano-bridged metal nanoparticles comprised therein are used forsubstitution by and/or sequestering of radionuclide and/or metalcations.

Li and coworkers describe that beta-sitosterol had radioprotectiveeffects on irradiated thymocytes by regulating the intracellular redoxbalances (decrease of ROS, increase of antioxidant enzyme activity suchas SOD, catalase) (Li, 2007). The antioxidant effect was confirmed onirradiated rats by Moustafa and co-workers (Moustafa, 2017).

The foregoing publications are hereby incorporated by reference in theirentireties.

-   -   Research progress in the radioprotective effect of superoxide        dismutase. Huang X J, Song C X, Thong C Q, Wang F S. Drug Discov        Ther. 2012 August; 6 (4):169-77.    -   Manganese superoxide dismutase, MnSOD and its mimics. Miriyala        S, Spasojevic I, Tovmasyan A, Salvemini D, Vujaskovic Z, St        Clair D, Batinic-Haberle I. Biochim Biophys Acta. 2012 May; 1822        (5):794-814    -   AEOL 10150 Mitigates Radiation-Induced Lung Injury in the        Nonhuman Primate: Morbidity and Mortality are Administration        Schedule-Dependent. MacVittie T J, Gibbs A, Farese A M, Barrow        K, Bennett A, Taylor-Howell C, Kazi A, Prado K, Parker G,        Jackson W III. Radiat Res. 2017 March; 187 (3):298-318.    -   Epperly M W, Dixon T, Wang H, Schlesselman J, Franicola D,        Greenberger J S. Radiat Res. 2008 October; 170 (4):437-43.    -   Antioxidant-chemoprevention diet ameliorates late effects of        total-body irradiation and supplements radioprotection by        MnSOD-plasmid liposome administration. Epperly M W, Wang H,        Jones J A, Dixon T, Montesinos C A, Greenberger J S. Radiat Res.        2011 June; 175 (6):759-65.    -   Manganese chloride treatment does not protect against acute        radiation injury of skin or crypt cells. Murata R, Nishimura Y,        Hiraoka M, Abe M, Satoh M. Radiat Res. 1995 September; 143        (3):316-9.    -   Toxicological Profile for Manganese. Williams M, Todd G D, Roney        N, Crawford J, Coles C, McClure P R, Garey J D, Zaccaria K,        Citra M. Atlanta (GA): Agency for Toxic Substances and Disease        Registry (US); 2012 September.    -   Beta-sitosterol decreases irradiation-induced thymocyte early        damage by regulation of the intracellular redox balance and        maintenance of mitochondrial membrane stability. Li CR, Zhou Z,        Lin R X, Thu D, Sun Y N, Tian L L, Li L, Gao Y, Wang S Q. J Cell        Biochem. 2007 October 15; 102 (3):748-58.    -   Beta-sitosterol upregulated paraoxonase-1 via peroxisome        proliferator-activated receptor-γ in irradiated rats. Moustafa E        M, Thabet N M. Can J Physiol Pharmacol. 2017 June; 95        (6):661-666.    -   US2014/0023701, US2017/0035909, U.S. Pat. Nos. 9,592,218 and        9,452,136, WO 2017/005899.

SUMMARY OF VARIOUS EMBODIMENTS

The disclosure provides for methods of treating a subject exposed toradiation and/or preventing radiation damage in a subject at risk forexposure to radiation comprising administering a reverse micellar systemthat comprises at least a sterol, 50 to 90% of acylglycerol, 1 to 20% oflecithin, ethanol, water, and optionally an active agent. In otherembodiments, the active agent comprises at least one manganese ion. Inother embodiments, the reverse micellar system does not include anucleic acid or a chelating agent. The disclosure provides for a reversemicellar system that comprises at least a sterol, 50 to 90% ofacylglycerol, 1 to 20% of lecithin, ethanol, water, and optionally anactive agent, for use in the treatment of a subject exposed to radiationand/or preventing radiation damage in a subject at risk for exposure toradiation.

The disclosure also provides for a reverse micellar system comprising atleast a sterol, 50 to 90% of acylglycerol, 1 to 20% of lecithin, ethanoland water, and wherein the reverse micellar system is free of metal andfree of a chelator. In other embodiments, the reverse micellar systemalso does not include a nucleic acid. In a particular embodiment, thereverse micellar system consists of at least a sterol, 50 to 90% ofacylglycerol, 1 to 20% of lecithin, ethanol and water. This reversemicellar system may be used in methods for treating a subject exposed toradiation and/or for preventing radiation damage in a subject at riskfor exposure to radiation.

It is an object of this disclosure to provide compositions for treatingand/or preventing cell, tissue or organ injury, in a living subject, dueto exposure to ionizing radiation without any side effects. It is alsoan object to provide compositions that are easy to administer to a largepopulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Post-TBI survival in CD1 Swiss mice after a 5.6 Gy doseirradiation at 0.16 Gy/min dose rate (LD_(50/30)). Treatments: (FIG.1A): Sample A: exemplary reverse micellar system and untreated; (FIG.1B): Sample B: manganese formulated at 500 μg/g in reverse micellarsystem and untreated; rm=radiomitigation;rpm=radioprotection/radiomitigation.

FIG. 2 : Post-TBI survival in CD1 Swiss mice after a 6.2 Gy doseirradiation at 0.16 Gy/min dose rate (LD_(90/30)). Treatments: Sample C:manganese formulated at 300 μg/g in reverse micellar system, Sample D:manganese formulated at 500 μg/g in reverse micellar system, anduntreated; rpm=radioprotection/radiomitigation.

FIG. 3 : Post-TBI survival in CD1 Swiss mice after a 7 Gy doseirradiation at 0.16 Gy/min dose rate (LD_(99.9/30)). Treatments: SampleE: exemplary reverse micellar system, Sample F: manganese formulated at500 μg/g in reverse micellar system, and untreated;rpm=radioprotection/radiomitigation.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

The disclosure provides a reverse micellar system comprising at least asterol, 50 to 90% of acylglycerol, 1 to 20 (e.g., 5 to 15% of lecithin),ethanol and water, wherein the weight ratio of lecithin to acylglycerolis from 0.05 to 0.4. The reverse micellar system may further comprise anactive agent, such as manganese. The reverse micellar system may be usedin the treatment or prevention of a cell, tissue or organ injury in aliving subject due to the exposure to ionizing radiation. For example,the disclosure provides a method of treating radiation damage in asubject (e.g., exposed to radiation therapy for treatment of a disease)comprising administering to the subject a reverse micellar systemdescribed herein in an amount effective to reduce radiation-induceddamage.

Reverse Micellar System

The reverse micellar system is generally a microemulsion comprising adispersion of water-nanodroplets in oil. The dispersion is stabilized bytwo surfactants (acylglycerol, more preferably a diacylglycerol andlecithin) and a co-surfactant (ethanol) that are most likely at thewater/oil interface. The water forms the internal phase and thehydrophobic tails of the lipids form the continuous phase. Reversemicelles containing oil(s), surfactant(s), co-surfactant(s), and anaqueous phase are also characterized as water-in-oil microemulsions.These microemulsions are thermodynamically stable and visually limpid.

In some embodiments, the reverse micellar system is a reverse phasesystem comprising an aqueous phase dispersed in an oil phase. Thereverse-phase system may comprise reverse or reverse swollen micelles,but these may be organized in higher ordered isotropic structures suchas water-in-oil microemulsion or anisotropic structures such as cubic,hexagonal, lamellar organizations.

Generally, the size of micelles is very small, such as less than 10 nm,less than 8 nm, or less than 6 nm. The size may vary with the quantityof added water and lecithin. The present invention relates moreparticularly to reverse micelles with an aqueous core of around 4 nm,preferably 3 to 5 nm, more preferably from 3.5 to 5 nm, in particularfrom 3.7 to 4.5 nm.

The ratios of the lipidic constituents (including sterol, acylglyceroland lecithin) in the reverse micellar system according to the inventioncan vary. For instance, the weight ratio sterol/acylglycerol can rangefrom 0.01 to 0.1, more particularly from 0.03 to 0.07. The weight ratiolecithin/acylglycerol can range from 0.05 to 0.40, in particular from0.06 to 0.25.

Preparation of the Reverse Micellar Systems

The reverse micellar systems described herein may be prepared by anytechnique known in the art. They are more particularly obtainable by thefollowing method:

-   -   (a) contacting (i) lecithin, (ii) ethanol, (iii) sterol, (iv)        acylglycerol, preferably diacylglycerol and (v) water,        preferably purified water, and    -   (b) stirring mixture obtained in step (a), at 40° C. or less,        and for a time sufficient to obtain formation of the reverse        micellar system.

The reverse micellar system described herein that further comprises anactive agent may be prepared by any technique known in the art. They aremore particularly obtainable by the following method:

-   -   (a) contacting (i) lecithin, (ii) ethanol, (iii) sterol, (iv)        acylglycerol, preferably diacylglycerol, (v) water, preferably        purified water, and (vi) at least one active agent, and    -   (b) stirring mixture obtained in step (a), at 40° C. or less,        and for a time sufficient to obtain formation of the reverse        micellar system.

The parameters of stirring (e.g., duration and speed of mechanicalstirring) can be readily determined by any one skilled in the art anddepend on experimental conditions. In practice, these parameters aresuch that a homogenous reverse micellar system is obtained; the speed isdetermined so as to enable formation of a visually limpid formulation,and duration of the stirring is such that the stirring may be stopped afew minutes after obtaining the visually limpid formulation.

Components of the Reverse Micellar Systems

Lecithin

The term lecithin refers to phosphatidylcholine. Phosphatidylcholine isalso known as 1,2-diacyl-glycero-3-phosphocholine or PtdCho. It iscomposed of a choline, a phosphate group, a glycerol and two fattyacids. It is a group of molecules, wherein the fatty acid compositionsvary from one molecule to another. Phosphatidylcholine may be obtainedfrom commercial lecithin that contains phosphatidylcholine in weightfractions from 20 to 98%. Preferably, the lecithin comprises more than92% weight phosphatidylcholine. The lecithin used in the examples isLIPOID S100 (sold by lipoid company) and contains phosphatidylcholine ata fraction of more than 94%.

As mentioned above, the ratios of the lipidic constituents (lecithin,sterol and acylglycerol) in the reverse micelle system can vary. Theweight ratio lecithin/acylglycerol can range from 0.05 to 0.40, inparticular from 0.06 to 0.25. The weight of lecithin corresponds to thetotal weight of the mixture containing phosphatidylcholine, such asLIPOID S100 described above.

Sterols

The sterols useful for the preparation of the reverse micelle systemsdescribed herein are preferably natural sterols, such as cholesterol orphytosterols (vegetable sterols). Sitosterol and cholesterol are thepreferred sterols useful for the reverse micelle systems. Preferably,the reverse micellar system comprises sitosterol, such beta-sitosterol.

Sitosterol and cholesterol are commercially available. Moreparticularly, commercial sitosterol, which is extracted from a varietyof pine called tall oil, can be used. In such a product, the sitosterolgenerally represents from 70 to 80% by weight of the product and isgenerally found in a mixture with campesterol and sitostanol inrespective proportions in the order of 10% each. Commercial sitosterolwhich is extracted from soya can also be used.

Preferably, the weight ratio sterol/acylglycerol can range from 0.01 to0.1, more particularly from 0.03 to 0.07. The weight of sterolcorresponds to the total weight of sterols used in the formulation.

Acylglycerols

Acylglycerols useful for the preparation of the reverse micellar systemsdescribed herein can be isolated from the majority of animals and morepreferably plants. Acylglycerols include mono-di and triacylglycerols.In a particular embodiment, acylglycerols preferentially used have thefollowing formula (I):

in which:

-   -   R1 is an acyl residue of a linear or branched, saturated or        unsaturated fatty acid having between 14 and 24 carbon atoms;    -   R₂ is an acyl residue of a linear or branched unsaturated fatty        acid having between 2 and 18 carbons atoms, or a hydrogen atom;    -   R3 is an acyl residue of a linear or branched, saturated or        unsaturated fatty acid having between 14 and 24 carbon atoms, or        a hydrogen atom.

According to a particular embodiment, R1 or R3, preferably only one ofR1 and R3, in particular only R1, represents an acyl residue of oleicacid (C18: 1[cis]-9).

According to a particular aspect, R2 has advantageously 18 carbon atoms,preferably R2 is an oleic acid residue (oleoyl group), one of itspositional isomers with respect to the double bond (cis-6, 7, 9, 11 and13) or one of its iso-branched isomers.

According to another particular aspect, R1 represents an oleoyl group.

According to another particular aspect, R3 is a hydrogen atom.

According to another particular aspect, R2 and R3 are hydrogen atoms.

As a general rule, oil containing a high concentration of oleic acidwill be chosen as a useful source of acylglycerols according to theinvention. Such oil usually contains a high proportion of acylglycerolsuseful according to the invention.

According to a particular aspect of the invention, the preferreddiglycerols of fatty acids are 1,2-dioleoylglycerol (or also namedherein 1,2-diolein) and 1-oleoyl-2-acetyl glycerol.

Acylglycerols are commercially available. For instance, glycerolmonooleate 40 contains about 32 to 52% of monoacylglycerol, 30 to 50% ofdiacylglycerol, 5 to 20% of triacylglycerol and is pharmaceuticallyaccepted (European Pharmacopeia (9th Edition), USP 25/NF20, and JapaneseStandard of food Additives).

Such product is commercially available by Gattefossé Company under thename Peceol®. In particular, Peceol® may comprise around 43 wt % ofmonoacylglycerol, around 44 wt % of diacylglycerol and around 11 wt % oftriacyl glycerol (the acyl fraction of Peceol® is mainly made ofoleoyl—usually around 80% of the acyl residue is oleoyl fraction).

The weight of acylgylycerol corresponds to the total weight of themixture containing an acylglycerol, or a mixture of acylglycerols, withglycerol and fatty acids derived from said acylglycerol(s).

Ethanol

Ethanol is generally an ethanol-water solution, wherein the ethanolamount is from about 90% to 99% by volume. In another embodiment,ethanol is absolute or anhydrous alcohol (that refers to ethanol with alow water content). There are various grades with maximum water contentsranging from 1% to a few parts per million (ppm) levels. Absoluteethanol is preferred.

Water

The water useful for the preparation of the reverse micelle systemsdescribed herein is preferably purified water; more particularlydistilled or deionized water.

Other Components

The reverse micellar systems described herein may comprise any type ofadditional components, such as alcohols other than ethanol.

The reverse micellar systems described herein may comprise at least onealcohol in addition to ethanol as defined above. The alcohols that maybe used are preferably linear or branched mono-alcohols with two to fourcarbons atoms. Examples of alcohols are 1-propanol, 2-propanol,2-methyl-1-propanol, isopropanol, and any mixture thereof. Polyols thatmay be used according to the invention are preferably glycerol andpropylene glycol.

The amounts of the components of the reverse micellar system can beadapted depending on the desired properties for the system, such asvisual appearance, viscosity, and/or concentration of active agent forinstance.

In some embodiments, the amounts of the components of the reversemicellar system are adjusted so that the reverse micellar system is inthe form of a liquid. A person of skill in the art can adapt therelative amounts of acylglycerol, sterol, lecithin, ethanol and water inthe reverse micellar system for obtaining a liquid with the desiredproperties, such as visual appearance, viscosity, and/or concentrationof active agent for instance.

Examples of amounts for the different components of the reverse-micellarsystems are the following:

-   -   The reverse-micellar system may comprise from 1 to 20%,        preferably 5 to 15% of lecithin.    -   The reverse-micellar system may comprise from 1 to 15%,        preferably from 5 to 15% water.    -   The reverse-micellar system may comprise from 5 to 15% alcohols,        including ethanol.    -   The reverse-micellar system may comprise from 0.825 to 5%        sterol.    -   The reverse-micellar system may comprise from 50 to 90%        acylglycerol.

Unless otherwise specified, the percentage values used herein are weightpercentages with respect to the total weight of the reverse-micellarsystem. The amounts specified herein will be adapted as to correspondmore specifically to the weight ratios identified above.

In some embodiments, the reverse micellar system does not compriseliposomes, nucleic acids, and/or chelating agents.

Active Agent

In some embodiments, the reverse micellar systems described herein mayoptionally comprise an active agent. The active agent is a compoundcapable of preventing and/or treating damage caused by the exposure tothe ionizing radiation of any type and capable of increasing theefficiency of reverse micellar system per se.

In other embodiments, the active agent is not used for its radionuclideor metal chelating/sequestering properties. For example, in someembodiments, the reverse micellar systems described herein are free ofthe chelating/sequestering agents disclosed in U.S. Pub. No.2017/0035909, hereby incorporated by reference in its entirety. Forexample, in some embodiments, the reverse micellar systems describedherein are free of DTPA, bisphosphonates, Prussian blue, EDTA,Trientine, D-penicillamine, Deferoxamine, BAL, DMSA, DMPS, Phytic acid,Hydroxypyridonates (HOPO), mercaptoacetyltriglycine (MAG3), chelatingpeptides, derivatives thereof and combinations thereof.

In other embodiments, the reverse micellar systems described herein arefree of the nucleic acids (e.g., DNA, RNA) described in U.S. Pat. No.9,452,136, hereby incorporated by reference in its entirety.

In other embodiments, the reverse micellar systems described herein arefree of the cyano-bridged metal nanoparticles described in WO2017/005899, hereby incorporated by reference in its entirety.

In an embodiment, the active agent may be a compound unknown in theprior art to be useful for preventing and/or treating the damages causedby the exposure to the ionizing radiation of any type.

In another embodiment, the active agent may be a compound known in theprior art for preventing and/or treating the damages caused by theexposure to the ionizing radiation of any type but not used because ofits side effects.

In some embodiments, the active agent comprises one or more ofmanganese, lithium, selenium, copper and/or zinc. In particularembodiments, the active agent is a pharmaceutically acceptable salt ofmanganese, such as manganese sulfate; a pharmaceutically acceptable saltof lithium, such as lithium citrate; a pharmaceutically acceptable saltof selenium, such as selenite sodium or selenite sulfate; apharmaceutically acceptable salt of copper such as copper sulfate;and/or a pharmaceutically acceptable salt of zinc, such as zinc sulfate.In other particular embodiments, the reverse micellar systems describedherein comprise at least 300 μg/g of manganese, or at least 500 μg/g ofmanganese; at least 600 μg/g of lithium; at least 100 μg/g of selenium,at least 100 μg/g of copper; and/or at least 500 μg/g of zinc. In aparticular embodiment, the reverse micellar systems described hereincomprise from 1,000 to 2,000 μg/g of manganese. The skilled person willbe able to adapt the ratios of the components of the reverse micellarsystem and the amount of active agent to encapsulate any active agentinto the reverse micellar system as described herein.

Subject

“Subject” refers to an organism to which the reverse micellar systemsdescribed herein can be administered. The subject may be a human or anon-human animal, such as a mammal.

Treatment

The disclosure contemplates various methods of treatment. For example,the disclosure provides methods of treating a subject from radiationdamage comprising administering an effective amount of a reversemicellar system described herein to a subject who has been exposed toradiation.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. The phrase “treating a disease” refers to inhibitingthe full development of a disease, for example, in a subject who is atrisk for a disease such as cancer.

In some embodiments, the treatment inhibits radiation damage in asubject. In other embodiments, an effective amount of a reverse micellarsystem described herein is administered to a subject exposed toradiation therapy for treatment of cancer. In particular embodiments,the cancer cells are more sensitive to the radiation therapy thannon-cancerous cells. In other embodiments, an effective amount of areverse micellar system described herein is administered to a subjectexposed to radioisotopes for medical diagnosis. In other embodiments, aneffective amount of a reverse micellar system described herein isadministered to a subject after military combat. In other embodiments,an effective amount of a reverse micellar system described herein isadministered to a subject after a nuclear attack or accident. In otherembodiments, an effective amount of a reverse micellar system describedherein is administered to a subject exposed to radiation but beforesymptoms of radiation damage appear.

Prevention

The disclosure contemplates various methods of prevention. For example,the disclosure provides methods of preventing a subject from radiationdamage comprising administering an effective amount of a reversemicellar system to a subject at risk to radiation exposure.

“Preventing” a disease or condition refers to prophylactic administeringa composition to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping a pathology or condition, or diminishing the severity of apathology or condition.

In some embodiments, an effective amount of a reverse micellar systemdescribed herein is administered to a subject at risk to radiationexposure. In other embodiments, an effective amount of a reversemicellar system described herein is administered to a subject beforeradiation therapy (e.g., for treatment of cancer). In particularembodiments, following radiation therapy for treatment of cancer, cancercells are more sensitive to the radiation therapy than non-cancerouscells. In other embodiments, an effective amount of a reverse micellarsystem described herein is administered to a subject beforeadministering radioisotopes for medical diagnosis. In other embodiments,an effective amount of a reverse micellar system described herein isadministered to a subject before the subject enters military combat. Inother embodiments, an effective amount of a reverse micellar systemdescribed herein is administered to a subject before a nuclear attack oraccident.

Dosage Regimen

The reverse micellar systems described herein are capable of beingabsorbed through mucosa. The reverse micellar system may be administeredvia different routes, including a transmucosal route, through buccalmucosal tissue, or permucosally.

As used herein, the terms “mucosa” and “mucosal” refer to a mucoustissue such as of the respiratory, digestive, or genital tissue.“Transmucosal delivery”, “mucosal delivery”, “mucosal administration”and analogous terms as used herein refer to the administration of acomposition through a mucosal tissue. “Transmucosal delivery”, “mucosaldelivery”, “mucosal administration” and analogous terms include, but arenot limited to, the delivery of a reverse micellar system describedherein via bronchi, gingival, lingual, nasal, oral, buccal, oesophageal,vaginal, rectal, and gastro-intestinal mucosal tissue.

The reverse micellar systems described herein may be administered at anytime respect to the radiation exposure. In some embodiments, the reversemicellar systems described herein are administered before exposure toionizing radiation, such as days before exposure to ionizing radiation.In other embodiments, the reverse micellar systems described herein areadministered after exposure to ionizing radiation, such as in the firstday, in the first hour, or in the first 15 minutes, following theexposure to ionizing radiation. In other embodiments, the reversemicellar systems described herein are administered more than 24 hours,more than 48 hours, or more than 96 hours after the end of the exposureto the ionizing radiation. In other embodiments, the reverse micellarsystems described herein may be continued to be administered for severaldays or weeks after the end of the exposure to the ionizing radiation.In other embodiments, the reverse micellar systems described herein areadministered before and after exposure to ionizing radiation.

As used herein, the terms radioprotection or radioprotector (rp) referto reverse micellar systems administered prior to radiation exposure.

As used herein, the terms radiomitigation or radiomitigator (rm) referto reverse micellar systems administered after the radiation exposure.

As used herein, the terms radioprotection/radiomitigation (rpm) refer toreverse micellar systems administered before and after the radiationexposure.

The skilled person will be able to adapt the number of dailyadministrations of reverse micellar system, the amount to beadministered, the frequency of administration and/or the moment when thetreatment is started or stopped in view of the type and intensity of theirradiation.

The skilled person will be able to adapt the number of dailyadministrations of reverse micellar system comprising an active agent,the amount to be administered, the frequency of administration and/orthe moment when the treatment is started or stopped in view of the typeand intensity of the irradiation and the amount of the active agentpresent in the reverse micellar systems.

The reverse micellar systems described herein may be formulated in acomposition further comprising a pharmaceutically acceptable support. Insome embodiments, a pharmaceutical composition comprising apharmaceutically acceptable support and a reverse micellar systemcomprising an acylglycerol, lecithin, ethanol, sterol water andoptionally an active agent is provided. The pharmaceutical compositionsmay be in the form of a capsule, a caplet, an aerosol, a spray, asolution or a soft elastic gelatin capsule.

The term “pharmaceutically acceptable support” refers to anypharmaceutically acceptable excipient, vehicle or carrier, well-known tothe person skilled in the art. Other additives well-known to the personskilled in the art such as stabilizers, drying agents, binders or pHbuffers may also be used in the pharmaceutical compositions describedherein. In particular embodiments, excipients that promote adherence ofthe finished product to the mucosa are included in the pharmaceuticalcompositions. In other embodiments, the reverse micellar system per seor comprising an active agent may be used in combination with one ormore additional agents.

Ionizing Radiation

The disclosure provides reverse micellar systems for treating and/orpreventing injury from radiation, such as ionizing radiation. Ionizingradiations relates to particles or waves having sufficiently high energyto ionize an atom or a molecule in a living body. Particle radiationsinclude alpha, beta or neutrons. Electromagnetic waves include X or γray.

As used herein, “radiation exposure” or “irradiation” refer to beingexposed or at risk to be exposed to ionizing radiations. Ionizingradiation may result from a number of sources including, but not limitedto, nuclear event, nuclear power plant accident, intentional terroristattack with a dirty bomb, medical practice such as diagnostic nuclearimaging and therapeutic radiations.

External and internal radiation exposure refer to the location of thesource of

radiation. “External ionizing radiation exposure” refers to the locationof the source outside of the body, whereas “internal ionizing radiationexposure” refers to the location of the source inside the body (e.g., aradionuclide inhaled, ingested or present into the bloodstream, such asafter injection or through wounds).

Ionizing Damage

The extent of radiation-induced internal damages will depend onduration, dose and type of radiation exposure and on the sensitivity ofdifferent tissues and organs. Radiation side effects can be classifiedas either acute, occurring during or immediately after radiationexposure, or late such as fibrosis, occurring weeks to months later.

As used here, “Acute Radiation Syndrome” or ARS relates to a damagecaused by whole-body or partial body irradiation in a human at high doseof penetrating radiation in a very short period of time (usually amatter of minutes). Clinical manifestations include gastrointestinal,hematopoietic, and neurovascular sub-syndromes.

In some embodiments, the radiation is potentially lethal. In otherembodiments, ionizing radiations induce sequential steps of cellular,tissue, organ, and total body injury. Injury and death of cells is acombination of direct and indirect ionizing radiation damages.

The exact mechanisms of reverse micellar systems described herein totreat radiation induced damage remain unclear. Without any intention ofbeing bound by any theory, one explanation is that reverse micellarsystems described herein act on the damaged cellular membranes in thewhole body. The active agent optionally comprised in the reversemicellar systems is delivered in the whole body and add their owntherapeutic activity at their target sites.

EXAMPLES

Various embodiments will now be particularly described by way ofexamples. The following descriptions of specific embodiments arepresented for purposes of illustration and description. They are notintended to be exhaustive of or to limit the disclosure.

Example 1: In Vivo Studies of the Efficacy of Reverse Micelles onSurvival of Irradiated mice at 30 Days Samples

Sample A

2.5 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 4.5 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 1.2 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 38.7 g of Peceol® wasadded thereto and magnetic stirring was carried out at 700 r/min at 37°C. to form an oil mixture. 0.4 g of purified water were added to 5.6 gof the oil mixture and stirred at room temperature few minutes to form areverse micellar system.

Sample B

0.5 g of purified water containing 13.9 mg of manganese sulfate (4.5 mgof metal manganese) was added to 8.5 g of the oil mixture described insample A and stirred at room temperature few minutes to form a reversemicellar system comprising 500 μg of metal manganese/g or 470 μg ofmetal manganese/ml (density of 0.94).

Sample C

0.3 g of purified water containing 5.6 mg of manganese sulfate (1.8 mgof metal manganese) was added to 5.7 g of the oil mixture described insample A and stirred at room temperature few minutes to form a reversemicellar system comprising 300 μg of metal manganese/g or 282 μg ofmetal manganese/ml (density of 0.94).

Sample D

0.4 g of purified water containing 9.3 mg of manganese sulfate (3.0 mgof metal manganese) was added to 5.6 g of the oil mixture described insample A and stirred at room temperature few minutes to form a reversemicellar system comprising 500 μg of metal manganese/g or 470 μg ofmetal manganese/ml (density of 0.94).

Sample E

10.0 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 18.0 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 5.0 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 155.0 g of Peceol® wasadded thereto and magnetic stirring was carried out at 700 r/min at 37°C. to form an oil mixture. 1.2 g of purified water was added to 18.8 gof the oil mixture and stirred at room temperature few minutes to form areverse micellar system.

Sample F

1.2 g of purified water containing 31.0 mg of manganese sulfate (10.0 mgof metal manganese) was added to 18.8 g of the oil mixture described insample E and stirred at room temperature few minutes to form a reversemicellar system comprising 500 μg of metal manganese/g or 470 μg ofmetal manganese/ml (density of 0.94).

Materials and Methods

3 studies, conducted in accordance with the European and Frenchregulations for animal experimentation (European directive 2010/63/EU,Sep. 22, 2010 and French decree 2013-118, Feb. 1, 2013), are describedbelow and in Table 1.

The animals used were male non-consanguineous Swiss CD1 mouse strain of4.5 to 6 weeks of age at receipt in the laboratory. Mice were allowedfree access to commercial diet (SSNIFF with 21% of raw proteins at thefirst step of life, and during experiment) distributed in food hoppers,and pre-filled acidified water bottles during acclimatization andexperimentation. 6 or 12 mice form a test batch.

Mice were exposed to a vertical X-ray beam (top to bottom), at 78-79 cmfrom the source (FSD), in a self-protected SARRP cabin (Small AnimalRadiation Research Platform, X-Strahl). They were kept vigilant, incontention in a box of Plexiglas, one individual per cage.

Total body irradiation (TBI) was calculated to be 50% (study 1), 90%(study 2) or 99.9% (study 3) lethal at day 30, i.e., LD_(50/30);LD_(90/30) or LD_(99.9/30). A “medium” dose rate of 0.16 Gy/min(programmed intensity of 3.2 mA) was chosen. The irradiation lastedabout 35 minutes and 54 seconds for a dose of 5.6 Gy output (study 1),about 39 minutes and 42 seconds for a dose of 6.2 Gy output (study 2)and about 44 minutes, for a dose of 7 Gy output (study 3). The animalsbeing held in contention, additional stress is to be taken intoconsideration, very probably growing as duration of restraint increases.

The treatments (Samples A to F) were administered to mice by rectalpermucosal route, using a micropipette with tips. Delivery was performedslowly in order to not hurt the animal and to cover the maximal mucosalsurface. Despite this precaution, the introduction of the tips into therectum can stimulate defecation. In case of any suspicion of loss ofproduct by defecation within 2 to 3 minutes following the rectalinjection, a second administration of the same amount (30 μl/mouse of 30g) was made. In practice, the vast majority of animals defecated, sothat the experimenter carried out a second intra rectal injection to allthe animals.

The treatment was started before irradiation and continued afterirradiation (rpm) or started after irradiation (rm).

The endpoints of the 3 studies were the percentage of mice survival at30 days

following TBI and the evolution of the survival curves during theintermediate period. The surviving mice were euthanized under isofluraneanaesthesia by decapitation at day 77 or 74 (study 1) or at day 30(studies 2 and 3) following irradiation exposure.

TABLE 1 Percent survival (%) RADIOPROTECTION RADIOMITIGATION Study 1Study 2 Study 3 Time of treatment (h, t0 − irradiation) LD_(50/30 d)LD_(90/30 d) LD_(99.9/30 d) Treatments H − 2 H − 1 H + 0.5 H + 2 H + 24H + 48 H + 96 n = 12/group n = 12/group n = 6/group No 58 treatmentSample A x x x x x 83 Sample B x x x x x 92 (Mn500) Sample B x x x 92(Mn500) No  8 treatment Sample C x x x 25 (Mn300) Sample D x x x 33(Mn500) No 0 treatment Sample E x x x x x x 0 Sample F x x x x x x 33 (Mn500)

Results

Study 1:

Mice (n=12/group) untreated or treated with sample A or B were exposedto a 5.6 Gy TBI (LD_(50/30)). Mice of group sample A were treated withthe reverse micellar system by rectal administration 1 and 2 hoursbefore TBI and 0.5, 2 and 24 hours after TBI. Sample B (manganeseformulated in reverse micelles) was administered depending on twoschedules: mice were treated by rectal administration 1 and 2 hoursbefore TBI and 0.5, 2 and 24 hours after TBI (group sample B rpm) orwere treated by rectal administration 0.5, 2 and 24 hours after TBI(group sample B rm). Under this protocol of irradiation, the survival ofuntreated animals reached 58% at day 30 and rose to 83% in animalstreated with sample A (see FIG. 1A). The survival of animals treatedwith sample B (groups sample B rpm and sample B rm) was 92%,irrespective of the schedule of treatment (see FIG. 1B).

Study 2:

Mice (n=12/group) untreated or treated were exposed to a 6.2 Gy TBI(LD_(90/30)). Mice were treated by rectal administration with sample Cor D following the same schedule: 1 and 2 hours before TBI and 24 hoursafter TBI. As shown in FIG. 2 , under this protocol of irradiation,untreated animals exhibited a survival of 8% and the treatment efficacyat 30 days was dependent on the dosage of manganese in the reversemicellar system: 300 μg/g in sample C or 500 μgig in sample D, with a25% and 33% survival respectively.

Study 3:

Mice (n=6/group) untreated or treated with sample E or F were exposed toa 7 Gy TBI (LD_(99.9/30)) Under this protocol of irradiation, micetreated by rectal administration with the reverse micellar system (groupsample E), 1 and 2 hours before TBI and 0.5, 24, 48, and 96 hoursfollowing TBI, exhibited a survival at day 30 of 0% such as untreatedgroup. However, during the intermediate period, the treatment withsample E extended the survival by 5 additional days in respect tountreated mice. Mice treated following the same schedule with manganeseformulated in reverse micelles (group sample F) exhibited a survival atday 30 of 33% (see FIG. 3 ).

Conclusions

The overall results (summarized in Table 1) show the effectiveness ofreverse micellar system per se or comprising manganese for increasingthe survival rate at 30 days of treated mice as compared with untreatedgroups.

The survival of animals is inversely proportional to the TBI dose, abetter efficacy being observed under the protocol of irradiation atLD_(50/30) compared to LD_(99.9/30) and LD_(90/30) at the same dose rate(0.16 Gy/min). At the highest TBI dose (LD_(99.9/30)), the addition ofmanganese in the reverse micellar system prolongs beyond 30 days itsintermediate effectiveness.

At LD_(50/30), the efficacy of the treatment with a reverse micellarsystem does not depend on its starting time (i.e. before or after theirradiation).

These results suggest that reverse micellar systems described herein maybe an effective medical countermeasure against severe and lethalradiation induced injuries.

Example 2: Formulations with High Concentrations of Manganese

Sample G

5.0 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 4.5 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 1.2 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 36.3 g of Peceol® wasadded thereto and magnetic stirring was carried out at 700 r/min at 37°C. to form an oil mixture.

0.2 g of purified water containing 12.5 mg of manganese sulfate (4.0 mgof metal manganese) was added to 3.8 g of the oil mixture and stirred atroom temperature few minutes to form a reverse micellar systemcomprising 1000 μg of metal manganese/g or 940 μg of metal manganese/ml(density of 0.94).

Sample H

6.0 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 4.5 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 1.2 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 33.7 g of Peceol® wasadded thereto and magnetic stirring was carried out at 700 r/min at 37°C. to form an oil mixture.

0.4 g of purified water containing 25.2 mg of manganese sulfate (8.0 mgof metal manganese) was added to 3.6 g of the oil mixture and stirred atroom temperature few minutes to form a reverse micellar systemcomprising 2000 μg of metal manganese/g or 1880 μg of metal manganese/ml(density of 0.94).

Sample I

0.4 g of purified water containing 37.7 mg of manganese sulfate (12.2 mgof metal manganese) was added to 3.6 g of the oil mixture described insample H and stirred at room temperature few minutes to form a reversemicellar system comprising 3000 μg of metal manganese/g or 2820 μg ofmetal manganese/ml (density of 0.94).

Sample J

3.8 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 2.2 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 0.6 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 15.4 g of Peceol® wasadded thereto and magnetic stirring was carried out at 700 r/min at 37°C. to form an oil mixture.

0.5 g of purified water containing 49.3 mg of manganese sulfate (16.0 mgof metal manganese) was added to 3.5 g of the oil mixture and stirred atroom temperature few minutes to form a reverse micellar systemcomprising 4000 μg of metal manganese/g or 3800 μg of metal manganese/ml(density of 0.95).

Example 3: Formulations with Other Active Agents

Sample K

10.0 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 18.0 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 5.0 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 155.0 g of Peceol® wasadded thereto and magnetic stirring was carried out at 700 r/min at 37°C. to form an oil mixture.

0.7 g of purified water containing 4.0 mg of selenite sulfate (1.2 mg ofmetal selenium) was added to 11.3 g of the oil mixture and stirred atroom temperature few minutes to form a reverse micellar systemcomprising 100 μg of metal selenium/g or 94 μg of metal selenium/ml(density of 0.94).

Sample L

94.0 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 84.6 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 23.5 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 647.9 g of Peceol® wasadded thereto and magnetic stifling was carried out at 700 r/min at 37°C. to form an oil mixture.

90.9 g of purified water containing 8.2 g of lithium citrate (0.6 g ofmetal lithium) was added to 850.0 g of the oil mixture and stirred atroom temperature few minutes to form a reverse micellar systemcomprising 638 μg of metal lithium/g or 600 μg of metal lithium/ml(density of 0.94).

Sample M

3.0 g of commercially available lecithin, containing more than 94% ofphosphatidylcholine, was dissolved in 5.4 g of absolute ethanol undermagnetic stirring at 300 r/min at room temperature. 1.5 g ofphytosterol, containing more than 70% of beta-sitosterol, was added tothe mixture and stirred in the same conditions. 46.5 g of Peceol® wasadded thereto and magnetic stirring was carried out at 700 r/min at 37°C. to form an oil mixture.

0.1 g of purified water containing 2.8 mg of zinc sulfate (1.0 mg ofmetal zinc) was added to 1.9 g of the oil mixture and stirred at roomtemperature few minutes to form a reverse micellar system comprising 500μg of metal zinc/g or 470 μg of metal zinc/ml (density of 0.94).

1-23. (canceled)
 24. A reverse micellar system comprising at least asterol, 50 to 90% of acylglycerol, 1 to 20% of lecithin, ethanol andwater, and the weight ratio of lecithin to acylglycerol is from 0.05:1to 0.4:1, wherein the reverse micellar system is free of a chelator,wherein the reverse micellar system is free of cyano-bridged metalnanoparticles, and wherein the reverse micellar system comprises atleast 300 μg/g of manganese.
 25. The reverse micellar system of claim24, comprising 5 to 15% lecithin.
 26. The reverse micellar system ofclaim 24, wherein the reverse micellar system is free of DTPA,bisphosphonates, Prussian blue, EDTA, Trientine, D-penicillamine,Deferoxamine, BAL, DMSA, DMPS, Phytic acid, Hydroxypyridonates (HOPO),mercaptoacetyltriglycine (MAG3), chelating peptides, derivatives thereofand combinations thereof.
 27. The reverse micellar system of claim 25,wherein the reverse micellar system is free of DTPA, bisphosphonates,Prussian blue, EDTA, Trientine, D-penicillamine, Deferoxamine, BAL,DMSA, DMPS, Phytic acid, Hydroxypyridonates (HOPO),mercaptoacetyltriglycine (MAG3), chelating peptides, derivatives thereofand combinations thereof.